Engineering the substrate and inhibitor specificities of human coagulation factor VIIa
Résumé
The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For factor VIIa (FVIIa), the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. Here, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning-substrate combinatorial libraries and evaluated the role of selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa showed P1 specificity exclusively towards Arg and Lys. In the S2 pocket Thr, Leu, Phe and Val were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed, and as a consequence, the optimal substrate for FVIIa could not be derived directly from a sub-site directed specificity screen. To evaluate the role of the active site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (c99), which is considered one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and reduced rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate factor X demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.
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